Emily Kistner-Griffin

Spoiling the Whole Bunch: Quality Control Aimed at Preserving the Integrity of High-Throughput Genotyping

False-positive or false-negative results attributable to undetected genotyping errors and confounding factors present a constant challenge for genome-wide association studies (GWAS) given the low signals associated with complex phenotypes and the noise associated with high-throughput genotyping. In the context of the genetics of kidneys in diabetes (GoKinD) study, we identify a source of error in genotype calling and demonstrate that a standard battery of quality-control (QC) measures is not sufficient to detect and/or correct it. We show that, if genotyping and calling are done by plate (batch), even a few DNA samples of marginally acceptable quality can profoundly alter the allele calls for other samples on the plate. In turn, this leads to significant differential bias in estimates of allele frequency between plates and, potentially, to false-positive associations, particularly when case and control samples are not sufficiently randomized to plates. This problem may become widespread as investigators tap into existing public databases for GWAS control samples. We describe how to detect and correct this bias by utilizing additional sources of information, including raw signal-intensity data.

Role for Prdx1 as a specific sensor in redox-regulated senescence in breast cancer

Recent studies suggest that Peroxiredoxin 1 (Prdx1), in addition to its known H2O2-scavenging function, mediates cell signaling through redox-specific protein–protein interactions. Our data illustrate how Prdx1 specifically coordinates p38MAPK-induced signaling through regulating p38MAPKα phosphatases in an H2O2 dose-dependent manner. MAPK phosphatases (MKP-1 and/or MKP-5), which are known to dephosphorylate and deactivate the senescence-inducing MAPK p38α, belong to a group of redox-sensitive phosphatases (protein tyrosine phosphatases) characterized by a low pKa cysteine in their active sites. We found that Prdx1 bound to both MKP-1 and MKP-5, but dissociated from MKP-1 when the Prdx1 peroxidatic cysteine Cys52 was over-oxidized to sulfonic acid, which in turn resulted in MKP-1 oxidation-induced oligomerization and inactivity toward p38MAPKα. Conversely, over-oxidation of Prdx1-Cys52 was enhancing in the Prdx1:MKP-5 complex with increasing amounts of H2O2 concentrations and correlated with a protection from oxidation-induced oligomerization and inactivation of MKP-5 so that activation toward p38MAPK was maintained. Further examination of this Prdx1-specific mechanism in a model of reactive oxygen species-induced senescence of human breast epithelial cells revealed the specific activation of MKP-5, resulting in decreased p38MAPKα activity. Taken together, our data suggest that Prdx1 orchestrates redox signaling in an H2O2 dose-dependent manner through the oxidation status of its peroxidatic cysteine Cys52.

Parent-of-Origin Effects of the Serotonin Transporter Gene Associated with Autism

A promoter‐linked insertion/deletion polymorphism of the serotonin transporter gene (SLC6A4) has been implicated in autism spectrum disorders (ASDs) in numerous family based association studies. However, the results of these investigations have been inconsistent in that both the long and short alleles have been shown to be over‐transmitted to affected offspring. In order to further elucidate the relationship between the 5‐HTTLPR variant and autism risk, we undertook a thorough study of parent‐of‐origin effects, maternal genotype effects, and offspring genotype effects in a sample of affected offspring from the Autism Genetic Resource Exchange (AGRE). Both the overall autism phenotype and measures of autism behaviors from the Autism Diagnostic Interview‐Revised [Lord et al. (1994); J Autism Dev Disord 24(5): 659–685] were considered. We found evidence of over‐transmission (risk allele short, P = 0.012), maternal effects (risk allele long, P = 0.035), and parent‐of‐origin effects (risk allele short from mother, P = 0.018) of the 5‐HTTLPR variant in the AGRE sample. Population‐ and gender‐specific effects were also explored as associations may be heterogeneous across populations and sexes. Parent‐of‐origin effects of the variant were associated with maternally inherited copies of the short allele that resulted in more impaired overall level of language (P = 0.04). Our study was conducted to further investigate the 5‐HTTLPR risk variants by identifying allelic associations that may be population‐specific, phenotype‐specific, or conferred by maternal or parent‐of‐origin effects. In light of conflicting observations from previous studies, these are just a few of the possible explanations that deserve attention.

Population differences in platinum toxicity as a means to identify novel genetic susceptibility variants.

Objectives Clinical studies show that Asians (ASN) are more susceptible to toxicities associated with platinum-containing regimens. We hypothesized that studying ASN as an ‘enriched phenotype’ population could enable the discovery of novel genetic determinants of platinum susceptibility. Methods Using well-genotyped lymphoblastoid cell lines from the HapMap, we determined cisplatin and carboplatin cytotoxicity phenotypes (IC50s) for ASN, Caucasians (CEU), and Africans (YRI). IC50s were used in genome-wide association studies. Results ASN were most sensitive to platinums, corroborating clinical findings. ASN genome-wide association studies produced 479 single-nucleotide polymorphisms (SNPs) associating with cisplatin susceptibility and 199 with carboplatin susceptibility (P<10−4). Considering only the most significant variants (P<9.99×10−6), backwards elimination was then used to identify reduced-model SNPs, which robustly described the drug phenotypes within ASN. These SNPs comprised highly descriptive genetic signatures of susceptibility, with 12 SNPs explaining more than 95% of the susceptibility phenotype variation for cisplatin, and eight SNPs approximately 75% for carboplatin. To determine the possible function of these variants in ASN, the SNPs were tested for association with differential expression of target genes. SNPs were highly associated with the expression of multiple target genes, and notably, the histone H3 family was implicated for both drugs, suggesting a platinum-class mechanism. Histone H3 has repeatedly been described as regulating the formation of platinum-DNA adducts, but this is the first evidence that specific genetic variants might mediate these interactions in a pharmacogenetic manner. Finally, to determine whether any ASN-identified SNPs might also be important in other human populations, we interrogated all 479/199 SNPs for association with platinum susceptibility in an independent combined CEU/YRI population. Three unique SNPs for cisplatin and 10 for carboplatin replicated in CEU/YRI. Conclusion Enriched ‘platinum susceptible’ populations can be used to discover novel genetic determinants governing interindividual platinum chemotherapy susceptibility.

Sequential ofatumumab and lenalidomide for the treatment of relapsed and refractory chronic lymphocytic leukemia and small lymphocytic lymphoma

Abstract Ofatumumab is a fully human anti-CD20 monoclonal antibody with enhanced antibody dependent and complement dependent cytotoxicity. Lenalidomide induces T cell and natural killer (NK) cell activation and in vitro enhances clearance of chronic lymphocytic leukemia (CLL) cells by monoclonal antibodies. We performed a multi-center, phase 2 trial of sequential treatment with ofatumumab and lenalidomide in patients with advanced, relapsed and refractory (R/R) CLL, consisting of ofatumumab 2000 mg intravenously on day 1 and lenalidomide 10 mg on days 8–28, for up to six cycles. Twenty-one subjects were included with median age of 63 years and two prior lines of therapy. The overall response rate was 47.6% and 23.8% had stable disease. Median overall survival was 21.5 months. Neutropenia and thrombocytopenia were the most frequent adverse events. Tumor flare reaction occurred in 43% of subjects. Intracycle sequential ofatumumab plus lenalidomide is active in high-risk R/R CLL and well tolerated except for frequent cytopenias.

Identification of a Variant in KDR Associated with Serum VEGFR2 and Pharmacodynamics of Pazopanib

Purpose: VEGF receptor (VEGFR) kinases are important drug targets in oncology that affect function of systemic endothelial cells. To discover genetic markers that affect VEGFR inhibitor pharmacodynamics, we performed a genome-wide association study of serum soluble vascular VEGFR2 concentrations [sVEGFR2], a pharmacodynamic biomarker for VEGFR2 inhibitors. Experimental Design: We conducted a genome-wide association study (GWAS) of [sVEGFR2] in 736 healthy Old Order Amish volunteers. Gene variants identified from the GWAS were genotyped serially in a cohort of 128 patients with advanced solid tumor with baseline [sVEGFR2] measurements, and in 121 patients with renal carcinoma with [sVEGFR2] measured before and during pazopanib therapy. Results: rs34231037 (C482R) in KDR, the gene encoding sVEGFR2 was found to be highly associated with [sVEGFR2], explaining 23% of the variance (P = 2.7 × 10−37). Association of rs34231037 with [sVEGFR2] was replicated in 128 patients with cancer with comparable effect size (P = 0.025). Furthermore, rs34231037 was a significant predictor of changes in [sVEGFR2] in response to pazopanib (P = 0.01). Conclusion: Our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identification of candidate pharmacogenetic markers. Genotyping for germline variants in KDR may have clinical utility in identifying patients with cancer with unusual sensitivity to effects of VEGFR2 kinase inhibitors. Clin Cancer Res; 21(2); 365–72. ©2014 AACR.

Loci nominally associated with autism from genome-wide analysis show enrichment of brain expression quantitative trait loci but not lymphoblastoid cell line expression quantitative trait loci

BackgroundAutism spectrum disorder is a severe early onset neurodevelopmental disorder with high heritability but significant heterogeneity. Traditional genome-wide approaches to test for an association of common variants with autism susceptibility risk have met with limited success. However, novel methods to identify moderate risk alleles in attainable sample sizes are now gaining momentum.MethodsIn this study, we utilized publically available genome-wide association study data from the Autism Genome Project and annotated the results (P <0.001) for expression quantitative trait loci present in the parietal lobe (GSE35977), cerebellum (GSE35974) and lymphoblastoid cell lines (GSE7761). We then performed a test of enrichment by comparing these results to simulated data conditioned on minor allele frequency to generate an empirical P-value indicating statistically significant enrichment of expression quantitative trait loci in top results from the autism genome-wide association study.ResultsOur findings show a global enrichment of brain expression quantitative trait loci, but not lymphoblastoid cell line expression quantitative trait loci, among top single nucleotide polymorphisms from an autism genome-wide association study. Additionally, the data implicates individual genes SLC25A12, PANX1 and PANX2 as well as pathways previously implicated in autism.ConclusionsThese findings provide supportive rationale for the use of annotation-based approaches to genome-wide association studies.

A GA microsatellite in the Fli1 promoter modulates gene expression and is associated with systemic lupus erythematosus patients without nephritis

IntroductionThe transcription factor Fli1 is implicated in the pathogenesis of systemic lupus erythematosus (SLE). Recently, a GAn polymorphic microsatellite was characterized in the mouse Fli1 promoter that modulates promoter activity and is truncated in two lupus mouse models compared to non-autoimmune prone mice. In this work, we characterize a homologous GAn microsatellite in the human Fli1 promoter. The purpose of this study is to determine the effect of the microsatellite length on Fli1 promoter activity in vitro and to determine if the length of the GAn microsatellite is associated with SLE and/or specific disease characteristics.MethodsConstructs with variable lengths of the GAn microsatellite in the Fli1 promoter were generated and analyzed in promoter/reporter (P/R) assays in a human T cell line. Using three SLE patient cohorts and matched controls, microsatellite length was measured and association with the presence of disease and the occurrence of specific disease manifestations was assessed.ResultsP/R assays demonstrated that the presence of a shorter microsatellite resulted in higher Fli1 promoter activity. A significant association was observed in the lupus cohort SLE in Gullah Health (SLEIGH) between the GA26 base pair allele and absence of nephritis.ConclusionsThis study demonstrates that a GAn microsatellite in the human Fli1 promoter is highly polymorphic. The length of the microsatellite is inversely correlated to Fli1 promoter activity in a human T cell line. Although no association between microsatellite length and lupus was observed, an association between a specific microsatellite length and patients without nephritis in the SLEIGH cohort was observed.

ETS1 transcriptional activity is increased in advanced prostate cancer and promotes the castrate-resistant phenotype

Advanced disease accounts for the majority of prostate cancer-related deaths and androgen deprivation therapy (ADT) is the standard of care for these patients. Many patients undergoing ADT become resistant to its effects and progress to castrate-resistant prostate cancer (CRPC). Current therapies for CRPC patients are inadequate, with progression-free survival rates as low as 2 months. The molecular events that promote CRPC are poorly understood. ETS (v-ets erythroblastosis virus E26 oncogene) transcription factors are regulators of carcinogenesis. Protein levels of the archetypical ETS factor, ETS1, are increased in clinical and latent prostate cancer relative to benign prostatic hyperplasia and normal prostate to promote multiple cancer-associated processes, such as energy metabolism, matrix degradation, survival, angiogenesis, migration and invasion. Our studies have found that ETS1 expression is highest in high-grade prostate cancer (Gleason 7 and above). Increased ETS1 expression and transcriptional activity promotes an aggressive and castrate-resistant phenotype in immortalized prostate cancer cells. Elevated AKT (v-akt murine thymoma viral oncogene homolog) activity was demonstrated to increase ETS1 protein levels specifically in castrate-resistant cells and exogenous ETS1 expression was sufficient to rescue invasive potential decreased by inhibition of AKT activity. Significantly, targeted androgen receptor activity altered ETS1 expression, which in turn altered the castrate-resistant phenotype. These data suggest a role for oncogenic ETS1 transcriptional activity in promoting aggressive prostate cancer and the castrate-resistant phenotype.

Genetic markers of immunoglobulin G and susceptibility to breast cancer.

Immunoglobulin GM allotypes, antigenic determinants of γ chains, are encoded by three very closely linked codominant genes on chromosome 14q32. Particular GM alleles/haplotypes are associated with antibody responses to certain tumor antigens and contribute to the cytotoxicity of breast cancer cells, but their possible role in susceptibility to this malignancy has not been adequately examined. Using a matched case-control design, we genotyped a large (1710 subjects) study population from Japan and Brazil for several GM alleles to determine whether these determinants are associated with susceptibility to breast cancer. After adjusting for the potential confounders, the GM 3 allele of IgG1 was significantly associated with susceptibility to breast cancer in white subjects from Brazil (OR=2.07, CI 1.16-3.71; p=0.0147). These data show that Caucasians with the GM 3 allele are over twice as likely to develop breast cancer as those who lack this allele. Since this allele modulates an immune evasion strategy of cytomegalovirus, the results also shed light on the possible mechanism underlying the reported involvement of this virus in the etiology of breast cancer.